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Parasitoids often exhibit high flexibility in their development depending on stages of their host at the parasitism, yet little is known about the mechanism underlying such flexibility. In the study, we evaluated the larval development time of the parasitoid Exorista sorbillans (Diptera: Tachinidae) on the lepidopteran model insect Bombyx mori (Lepidoptera: Bombycidae). We found that the development duration of E. sorbillans larvae parasitizing on the late-developmental silkworms was significantly shorter than that of the larvae parasitizing on the early-developmental hosts. Intriguingly, the 2nd-3rd instar molting of parasitoid always occurred when the ecdysteroid titers in the host were increased to higher levels. Furthermore, inhibiting the release of ecdysteroids to parasitic abdomen by thorax-abdomen ligation of the host only repressed the 2nd-instar growth and molting of E. sorbillans larvae, but had no effect on their pupation. Meanwhile, the ecdysone synthesis and 20-hydroxyecdysone (20 E) signaling in larval parasitoids were impeded after ligation treatment. Moreover, exogenous 20 E application could largely rescue the defect in 2nd instar growth and molting through stimulating ecdysone synthesis and signaling in E. sorbillans. Our results indicate that the parasitoid requires the host ecdysteroids to stimulate 20 E signaling and the subsequent 2nd-instar growth and molting. These findings will improve our understanding of the host utilization strategies of parasitoids, and contribute to the development of in vitro rearing procedures of tachinid parasitoids for biological control., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Microsporidia are opportunistic fungal-like pathogens that cause microsporidiosis, which results in significant economic losses and threatens public health. Infection of domesticated silkworms by the microsporidium Nosema bombycis causes pébrine disease, for which this species of microsporidia has received much attention. Research has been conducted extensively on this microsporidium over the past few decades to better understand its infection, transmission, host-parasite interaction, and detection. Several tools exist to study this species including the complete genome sequence of N. bombycis. In addition to the understanding of N. bombycis being important for the silkworm industry, this species has become a model organism for studying microsporidia. Research on biology of N. bombycis will contribute to the development of knowledge regarding microsporidia and potential antimicrosporidia drugs. Furthermore, this will provide insight into the molecular evolution and functioning of other fungal pathogens., (© 2024 International Society of Protistologists.)

The egg parasitoid Mesocomys trabalae Yao, Yang, and Zhao is used as a biocontrol agent against the emerging defoliator pest Caligula japonica Moore in East Asia. It has been proven that the eggs of Antheraea pernyi Guérin-Méneville can be used as a factitious host for the mass production of M. trabalae. We examined the parasitic behavior and morphological characteristics of the developmental stages of M. trabalae reared on A. pernyi eggs. The parasitic behavior of M. trabalae encompasses 10 steps, involving searching, antennation, locating the oviposition site, drilling, probing, detecting, oviposition, host feeding, grooming, and resting, with the oviposition step further divided into 3 stages. We determined that the parasitoid released an egg during the second stage of the oviposition step, while her body remained in a relatively static state. Among all the steps in parasitic behavior, probing occupied the longest time, accounting for 26.33% of the entire parasitism process. It was followed by oviposition (15.88%), drilling (15.10%), antennation (13.09%), detecting (10.79%), host feeding (10.02%), and the remaining steps, each occupying less than 5.00% of the total time in steps. The pre-emergence of adult M. trabalae comprised of 4 stages: egg (0-1 day), larva (2-6 days), prepupa (7-11 days), pupa (12-20 days), followed by the development into an adult, and it usually took 20-22 days to develop from an egg into an adult at 25°C. This study advances our understanding of the biology of Mesocomys parasitoids and their mass-rearing for use in augmentation programs., (© The Author(s) 2024. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Parasitoids are important components of the natural enemy guild in the biological control of insect pests. They depend on host resources to complete the development of a specific stage or whole life cycle and thus have evolved towards optimal host exploitation strategies. In the present study, we report a specific survival strategy of a fly parasitoid Exorista sorbillans (Diptera: Tachinidae), which is a potential biological control agent for agricultural pests and a pest in sericulture. We found that the expression levels of nitric oxide synthase (NOS) and nitric oxide (NO) production in host Bombyx mori (Lepidoptera: Bombycidae) were increased after E. sorbillans infection. Reducing NOS expression and NO production with an NOS inhibitor (NG-nitro-L-arginine methyl ester hydrochloride) in infected B. mori significantly impeded the growth of E. sorbillans larvae. Moreover, the biosynthesis of 20-hydroxyecdysone (20E) in infected hosts was elevated with increasing NO production, and inhibiting NOS expression lowered 20E biosynthesis. More importantly, induced NO synthesis was required to eliminate intracellular bacterial pathogens that presumably competed for shared host resources. Inhibiting NOS expression down-regulated the transcription of antimicrobial peptide genes and increased the number of bacteria in parasitized hosts. Collectively, this study revealed a new perspective on the role of NO in host-parasitoid interactions and a novel mechanism for parasitoid regulation of host physiology to support its development., (© 2024 Royal Entomological Society.)

Nosema bombycis, an obligate intracellular parasite, is a single-celled eukaryote known to infect various tissues of silkworms, leading to the manifestation of pebrine. Trehalase, a glycosidase responsible for catalyzing the hydrolysis of trehalose into two glucose molecules, assumes a crucial role in thermal stress tolerance, dehydration, desiccation stress, and asexual development. Despite its recognized importance in these processes, the specific role of trehalase in N. bombycis remains uncertain. This investigation focused on exploring the functions of trehalase 3 in N. bombycis (NbTre3). Immunofluorescence analysis of mature (dormant) spores indicated that NbTre3 primarily localizes to the spore membrane or spore wall, suggesting a potential involvement in spore germination. Reverse transcription-quantitative polymerase chain reaction results indicated that the transcriptional level of NbTre3 peaked at 6 h post N. bombycis infection, potentially contributing to energy storage for proliferation. Throughout the life cycle of N. bombycis within the host cell, NbTre3 was detected in sporoplasm during the proliferative stage rather than the sporulation stage. RNA interference experiments revealed a substantial decrease in the relative transcriptional level of NbTre3, accompanied by a certain reduction in the relative transcriptional level of Nb16S rRNA. These outcomes suggest that NbTre3 may play a role in the proliferation of N. bombycis. The application of the His pull-down technique identified 28 proteins interacting with NbTre3, predominantly originating from the host silkworm. This finding implies that NbTre3 may participate in the metabolism of the host cell, potentially utilizing the host cell's energy resources., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

In insects infections trigger hemocyte-mediated immune reactions including degranulation by exocytosis; however, involvement of mediator enzymes in degranulation process is unknown in insects. We report here that in silkworm Bombyx mori, infection by endoparasitoid Exorista bombycis and microsporidian Nosema bombycis activated granulation in granulocytes and promoted degranulation of accumulated structured granules. During degranulation the mediator lysosomal enzyme β-hexosaminidase showed increased activity and expression of β-hexosaminidase gene was enhanced. The events were confirmed in vitro after incubation of uninfected hemocytes with E. bombycis larval tissue protein. On infection, cytotoxicity marker enzyme lactate dehydrogenase (LDH) was released from the hemocytes illustrating cell toxicity. Strong positive correlation (R 2  = 0.71) between LDH activity and β-hexosaminidase released after the infection showed parasitic-protein-induced hemocyte damage and accompanied release of the enzymes. Expression of β-hexosaminidase gene was enhanced in early stages after infection followed by down regulation. The expression showed positive correlation (R 2  = 0.705) with hexosaminidase activity pattern. B. mori hexosaminidase showed 98% amino acid similarity with that of B. mandarina showing origin from same ancestral gene; however, 45-60% varied from other lepidopterans showing diversity. The observation signifies the less known association of hexosaminidase in degranulation of hemocytes induced by parasitic infection in B. mori and its divergence in different species.

Trichogramma leucaniae is believed to be an efficient biological control agent for controlling the soybean pod borer [SPB; Leguminivora glycinivorella]. The large eggs of Chinese oak silkworm, Antheraea pernyi, are one of the best alternative host for mass production of Trichogramma. However, they are considered poor host for the growth and development of T. leucaniae. Here, we determine the feasibility of successive rearings of T. leucaniae on the large eggs for eight generations and evaluated their capacity of parasitizing SPB eggs of different ages. In the first four generations, the suitability of T. leucaniae reared on large eggs exhibited a significant increasing tendency and then decreased with the successive generations thereafter. The percentage of parasitized eggs and number of emerged adults per egg were increased from 40.0% and 10.8 adults/egg in F1 generation to 86.7% and 36.4 adults/egg in F4 generation respectively. In addition, T. leucaniae reared on A. pernyi for four generations significantly parasitized more SPB eggs regardless of egg age compared with those reared on Corcyra cephalonica eggs. These results provided useful information on the feasibility of mass production of T. leucaniae by reared for successive generations on A. pernyi large eggs., Competing Interests: The authors have declared that no competing interests exist.

Invertebrates lack an adaptive immune response and thus are reliant on their innate immune response for eliminating invading pathogens. The innate immune responses of silkworms against the pathogen Nosema bombycis include: hemocyte aggregation, melanization, antimicrobial peptides, etc. In our current study, we discovered that a silkworm hemostasis-related protein, hemocytin, is up-regulated after Nosema bombycis infection. This novel finding lead to our hypothesis that hemocytin participates in immune responses against N. bombycis. We investigated this hypothesis by analyzing the adhesive effects of hemocytin to invading N. bombycis, and the hemocytin-mediated hemocyte aggregation and hemolymph melanization. We showed that hemocytin can adhere to the surface of N. bombycis, which facilitates the agglutination of N. bombycis and hemocytes as well as the subsequent melanization. Moreover, when we utilize RNAi technology to decrease in vivo hemocytin expression, we found that the proliferation of N. bombycis within the host significantly increased. These results support our hypothesis that hemocytin exerts pro-inflammatory effects by facilitating pathogen agglutination, along with hemocyte aggregation and melanization, to combat N. bombycis. Our study is the first to determine a function of hemocytin in innate immunity against N. bombycis. Moreover, our findings are of great importance to provide potential targets for developing novel strategy against microsporidia infection., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The secretion of hexokinases (HKs) by microsporidia followed by their accumulation in insect host nuclei suggests that these enzymes play regulatory and catalytic roles in infected cells. To confirm whether HKs exert catalytic functions in insect cells, we expressed in E. coli the functionally active HKs of two entomopathogenic microsporidia, Nosema bombycis and Nosema ceranae, that cause silkworm and honey bee nosematoses. N. bombycis HK with C-terminal polyHis tag and N. ceranae enzyme with N-terminal polyHis tag were cloned into pOPE101 and pRSET vectors, respectively, and overexpressed. Specific activities of N. bombycis and N. ceranae enzymes isolated by metal chelate affinity chromatography were 29.2 ± 0.5 and 60.2 ± 1.2 U/mg protein at an optimal pH range of 8.5-9.5. The kinetic characteristics of the recombinant enzymes were similar to those of HKs from other parasitic and free-living organisms. N. bombycis HK demonstrated Km 0.07 ± 0.01 mM and k cat 1726 min -1 for glucose, and Km 0.39 ± 0.05 mM and k cat 1976 min -1 for ATP, at pH 8.8. N. ceranae HK showed Km 0.3 ± 0.04 mM and k cat 3293 min -1 for glucose, and Km 1.15 ± 0.11 mM and k cat 3732 min -1 for ATP, at the same pH value. These data demonstrate the capability of microsporidia-secreted HKs to phosphorylate glucose in infected cells, suggesting that they actively mediate the effects of the parasite on host metabolism. The present findings justify further study of the enzymes as targets to suppress the intracellular development of silkworm and honey bee pathogens.

We reared a Telenomus species from eggs of Bombyx mandarina (Moore) (Lepidoptera: Bombycidae) and Bombyx mori (Linnaeus) (Lepidoptera: Bombycidae) in Japan, and from eggs of B. mandarina in Taiwan. Morphological examination revealed that this Telenomus species is new to science. In this article, we describe it as Telenomus moricolus Matsuo et Hirose, sp. nov. Because B. mandarina is considered to be an ancestor of B. mori, a domestic insect, it is reasonable to assume that B. mandarina is an original host of T. moricolus. This is the second discovery of an egg parasitoid attacking wild and domesticated silkworms, following the first discovery of T. theophilae, a Chinese species. The significance of the discovery of T. moricolus is discussed in relation to examining the effects of host-insect domestication on egg parasitism.

Background: Microsporidia are a group of eukaryotic intracellular parasites that infect almost all vertebrates and invertebrates. However, there is little information available of how microsporidia obtain nutrients and energy from host cells. The purpose of this study was to investigate the energy and material requirements of Nosema bombycis for the invasion procedure through analyzing the global variation of the gene expression, protein abundance, fatty acids level and ATP flux induced by the microsporidia N. bombycis infection in the midgut of the silkworm Bombyx mori., Methods: A suppression subtractive hybridization (SSH) and quantitative real-time PCR (qPCR) analysis were performed to identify the genes upregulated in the midgut of B. mori 48 h following N. bombycis infection. Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were used to annotate and summarize the differentially expressed genes, according to the categories 'molecular function', 'cellular component' and 'biological process'. To evaluate the nutrition material and energy costs in B.mori infected by N. bombycis, biochemical analysis was performed to determine the variation of protein abundance, fatty acid levels and ATP flux with or without the microsporidia N. bombycis infection in the midgut of the silkworm B. mori., Results: A total of 744 clones were obtained, 288 clones were randomly selected for sequencing, and 110 unigenes were generated. Amongst these, 49.21%, 30.16% and 14.29% genes were involved in 19 molecular functions, 19 biological processes and nine cellular components, respectively. A total of 11 oxidative phosphorylation- and eight proton-coupled ATP synthesis-related genes were upregulated. Seven protein degradation-, three fat degradation-related genes were upregulated, and no genes related to the de novo synthesis of amino acids and fatty acids were significantly upregulated. The data from the biochemical analysis showed the contents of total protein and ATP of B. mori midgut tissues decreased significantly, whereas the fatty acid content did not significantly change after four days of N. bombycis infection. Microsporidia N. bombycis infection upregulated the expression level of genes involved in host ATP synthesis, protein and fat degradation, which eventually causes the obvious decline of protein content and ATP synthesis in the host midgut, whereas the fatty acids content did not change significantly., Conclusions: This study suggested to some extent that N. bombycis invasion can activate the host protein degradation and accelerate the production of host ATP. Microsporidia of N. bombycis show preference for proteins rather than fatty acids from the host to ensure the material preparation required by their parasitic life-cycle. Requirements of N. bombycis for energy were also mainly dependent on the host ATP production. This study provides a new data that may help our understanding of the molecular mechanisms of obtaining energy and nutrients from the host by the microsporidium N. bombycis.

Microsporidia Nosema bombycis CQ1 can be vertically transmitted in silkworm Bombyx mori but Vairimorpha necatrix BM cannot. Therefore, the pathological differences in silkworm infected with these two microsporidia required clarification. Here, we compared the virulence of N. bombycis CQ1 and V. necatrix BM against silkworm. The pathological characteristics in intestine, testis and ovary were surveyed using paraffin sections, scanning electron microscopy and transmission electron microscopy. Our data firstly showed that the virulence of V. necatrix BM was weaker than that of N. bombycis CQ1. Secondly, the typical symptom of V. necatrix BM infection is making xenomas, which are full of pathogens in different stages, at the posterior of intestine. However, no xenomas were formed surrounding intestines infected with N. bombycis CQ1. Thirdly, N. bombycis CQ1 can cluster spores near the trachea while infecting ovaries. It is worth noting that N. bombycis CQ1 infected epithelial cells and connective tissues of ovaries, while V. necatrix BM did not. Although silkworm ovaries can not be infected by V. necatrix BM in vivo, it can infect embryonic and ovarian cell lines in vitro. This study is the first report about comparing infection features of N. bombycis CQ1 and V. necatrix BM in silkworm tissues and it provided elaborate and visual information of pathological characteristics which can help to explain the different transmission strategies of these two microsporidia., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Parasitization of silkworm, Bombyx mori by invasive larva of dipteran parasitoid Exorista bombycis caused upto 20% revenue loss in sericulture. The parasitism was successful by suppressing host immune system however mechanism of immune suppression induced by E. bombycis is unknown which is unravelled here. The infestation induced cytotoxic symptoms in host hemocytes, such as vacuolated cytoplasm, porous plasma membrane, indented nuclei with condensed chromatin and dilated RER. One of the markers of necrosis is cell permeabilization, which can be measured as released lactate dehydrogenase (LDH). LDH level showed significantly (P<0.01) high release into extracellular medium in vitro after exposure of hemocytes to parasitoid larval tissue protein compared with control revealing membrane permeability and loss of cell integrity. At five minutes after exposure, cytotoxicity was 43% and was increased to 99% at 3h. The cytotoxicity is signalled by increased content of hydrogen peroxide (H2O2) causing lipid peroxidation followed by porosity in plasma membrane. A test for lipid peroxidation by measurement of lipid peroxidation breakdown product, malondialdehyde (MDA) revealed significant increase in peroxidation from one to 24 h post-invasion, with maximum at 12 h (P<0.008). Level of reactive oxygen species measured as H2O2 production increased from 6 to 12 h post-invasion and continued to increase significantly (P<0.03) reaching maximum at 48 h. These observations reveal that dipteran endoparasitoid invasion induced H2O2 production in the hemocytes causing cytotoxicity, lipid peroxidation and membrane porosity that suppressed both humoral- and cell-mediated immune responses of hemocytes in B. mori.

Bracoviruses associate symbiotically with thousands of parasitoid wasp species in the family Braconidae, working as virulence gene vectors, and allowing the development of wasp larvae within hosts. These viruses are composed of multiple DNA circles that are packaged into infective particles, and injected together with wasp's eggs during parasitization. One of the viral segments of Cotesia vestalis bracovirus contains a gene that has been previously described as a helicase of unknown origin. Here, we demonstrate that this gene is a Rep/Helicase from an intact Helitron transposable element that covers the viral segment almost entirely. We also provide evidence that this element underwent at least two horizontal transfers, which appear to have occurred consecutively: first from a Drosophila host ancestor to the genome of the parasitoid wasp C. vestalis and its bracovirus, and then from C. vestalis to a lepidopteran host ( Bombyx mori ). Our results reinforce the idea of parasitoid wasps as frequent agents of horizontal transfers in eukaryotes. Additionally, this Helitron-bracovirus segment is the first example of a transposable element that effectively became a whole viral circle., (Copyright © 2017 Heringer et al.)

Hymenopteran parasitoids inject various factors including polydnaviruses along with their eggs into their host insects that suppress host immunity reactions to the eggs and larvae. Less is known about the mechanisms evolved in dipteran parasitoids that suppress host immunity. Here we report that the dipteran, Exorista bombycis, parasitization leads to pro-oxidative reactions and activation of anti-oxidative enzymes in the silkworm Bombyx mori larva. We recorded increased activity of oxidase, superoxide dismutase, thioredoxin peroxidase, catalase, glutathione-S-transferase (GST), and peroxidases in the hemolymph plasma, hemocytes, and fat body collected from B. mori after E. bombycis parasitization. Microarray and qPCR showed differential expression of genes encoding pro- and anti-oxidant enzymes in the hemocytes. The significance of this work lies in increased understanding of dipteran parasitoid biology., (© 2017 Wiley Periodicals, Inc.)

Nosema bombycis is an obligate intracellular parasitic fungus that utilizes a distinctive mechanism to infect Bombyx mori Spore germination can be used for host cell invasion; however, the detailed mechanism remains to be elucidated. The ricin-B-lectin (RBL) gene is significantly differentially regulated after N. bombycis spore germination, and NbRBL might play roles in spore germination and infection. In this study, the biological function of NbRBL was examined. Protein sequence analysis showed that NbRBL is a secreted protein that attaches to carbohydrates. The relative expression level of the NbRBL gene was low during the first 30 h post-infection (hpi) in BmN cells, and high expression was detected from 42 hpi. Gene cloning, prokaryotic expression, and antibody preparation for NbRBL were performed. NbRBL was detected in total and secreted proteins using western blot analysis. Subcellular localization analysis showed that NbRBL is an intracellular protein. Spore adherence and infection assays showed that NbRBL could enhance spore adhesion to BmN cells; the proliferative activities of BmN cells incubated with anti-NbRBL were higher than those in negative control groups after N. bombycis infection; and the treatment groups showed less damage from spore invasion. We therefore, propose that NbRBL is released during spore germination, enhances spore adhesion to BmN cells, and contributes to spore invasion., (© The Author 2016. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Green muscardine caused by Metarhizium rileyi affects sericulture, and is typically enzootic and occurs frequently at low incidence. We collected 152 M. rileyi isolates from silkworm cadavers in eight sericulture areas in seven provinces of China, and four strains from other Lepidoptera larvae in Qianshan(QS) County, Anhui province. Nine microsatellite inter-simple sequence repeat (ISSR) primers produced 91 distinct and reproducible bands, revealing a high level (90.11%) of DNA polymorphism. Unweighted Pair Group Method with Arithmetic Mean (UPGMA) cluster analysis divided the populations into four groups, with isolates from Qianshan County forming a single branch. All the 156 M. rileyi isolates were heterogenic and polyphyletic and did not displayed typical regional distribution except strains from Qianshan country. PCA analysis of the nine populations of M. rileyi revealed similar phylogenies among accessions. Genetic differentiation index (G st ) among eight enzootic populations was 0.3789 and gene flow (N m ) was 0.4098, suggesting the low gene flow maintained a high degree of differentiation. Gst between the enzootic population of Qianshan County and other seven populations exceeded the threshold of severe differentiation, with moderate differentiation between the remaining seven enzootic populations. Analysis of molecular variance (AMOVA) showed most ISSR variation (61%) among isolates occurred within populations. No significant correlation was observed between geographical and genetic distance. According to cluster analysis based on single enzootic population, every enzootic population showed dominance, namely mainly constituted of strains with high genetic similarity. These data indicated that the green muscardine in each local silkworm population was predominantly caused by a native group of M. rileyi. Furthermore, G st and N m of M. rileyi from silkworm and other Lepidoptera larvae in Qianshan County were 0.1174 and 1.8791, respectively, suggesting strains isolated from different hosts in Qianshan County do not show obvious host specificity. This demonstrated that host transfer may take place in silkworm and other insects., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Beauveria bassiana is a kind of world-wide entomopathogenic fungus and can also colonize plant rhizosphere. Previous researches showed differential expression of genes when entomopathogenic fungi are cultured in insect or plant materials. However, so far there is no report on metabolic alterations of B. bassiana in the environments of insect or plant. The purpose of this paper is to address this problem. Herein, we first provide the metabolomic analysis of B. bassiana cultured in insect pupae extracts (derived from Euproctis pseudoconspersa and Bombyx mori, EPP and BMP), plant root exudates (derived from asparagus and carrot, ARE and CRE), distilled water and minimal media (MM), respectively. Principal components analysis (PCA) shows that mycelia cultured in pupae extracts and root exudates are evidently separated and individually separated from MM, which indicates that fungus accommodates to insect and plant environments by different metabolic regulation mechanisms. Subsequently, orthogonal projection on latent structure-discriminant analysis (OPLS-DA) identifies differential metabolites in fungus under three environments relative to MM. Hierarchical clustering analysis (HCA) is performed to cluster compounds based on biochemical relationships, showing that sphingolipids are increased in BMP but are decreased in EPP. This observation further implies that sphingolipid metabolism may be involved in the adaptation of fungus to different hosts. In the meantime, sphingolipids are significantly decreased in root exudates but they are not decreased in distilled water, suggesting that some components of the root exudates can suppress sphingolipid to down-regulate sphingolipid metabolism. Pathway analysis finds that fatty acid metabolism is maintained at high level but non-ribosomal peptides (NRP) synthesis is unaffected in mycelia cultured in pupae extracts. In contrast, fatty acid metabolism is not changed but NRP synthesis is high in mycelia cultured in root exudates and distilled water. This indicates that fungal fatty acid metabolism is enhanced when contacting insect, but when in the absence of insect hosts NRP synthesis is increased. Ornithine, arginine and GABA are decreased in mycelia cultured in pupae extracts and root exudates but remain unchanged in distilled water, which suggests that they may be associated with fungal cross-talk with insects and plants. Trehalose and mannitol are decreased while adenine is increased in three conditions, signifying carbon shortage in cells. Together, these results unveil that B. bassiana has differential metabolic responses in pupae extracts and root exudates, and metabolic similarity in root exudates and distilled water is possibly due to the lack of insect components., (Copyright © 2015. Published by Elsevier Inc.)

Nosema bombycis, a pathogen of silkworm pebrine, is an obligate unicellular eukaryotic parasite. It is reported that the spore wall proteins have essential functions in the adherence and infection process of microsporidia. To date, the information related to spore wall proteins from microsporidia is still limited. Here, a 44 kDa spore wall protein NbSWP16 was characterized in N. bombycis. In NbSWP16, a 25 amino acids signal peptide and 3 heparin binding motifs were predicted. Interestingly, a region that contains 3 proline-rich tandem repeats lacking homology to any known protein was also present in this protein. The immunofluorescence analysis (IFA) demonstrated that distinct fluorescent signals were detected both on the surface of mature spores and the germinated spore coats. Immunolocation by electron microscopy revealed that NbSWP16 localized on the exospore regions. Finally, spore adherence analysis indicated that spore adherence to host cell was decreased more than 20% by anti-NbSWP16 blocking compared with the negative control in vitro. In contrast with anti-NbSWP16, no remarkable decrement inhibition was detected when antibodies of NbSWP16 and NbSWP5 were used simultaneously. Collectively, these results suggest that NbSWP16 is a new exospore protein and probably be involved in spore adherence of N. bombycis.

A new microsporidium was isolated from Histia rhodope Cramer (Lepidoptera, Zygaenidae), a pest of Bischofia javanica BL. in China. The morphology and molecular systematic of this novel microsporidian isolate had been described in this study. The spores were long oval and measured 3.1 × 1.9 μm on fresh smears. Ultrastructure of the spores was characteristic for the genus Nosema: 14-15 polar filament coils, posterior vacuole, and a diplokaryon. The sequenced rRNA gene of this isolate is 4309 bp long. The organization of the rRNA gene is 5'-LSU rRNA-ITS-SSU rRNA-IGS-5S-3', which is similar to that of other Nosema species (such as Nosema bombycis). Phylogenetic analysis based on LSU rRNA gene and SSU rRNA gene both revealed that this novel micorsporidian which isolated from H. rhodope had close relationship to the genus Nosema. Additionally, this isolate can also cause systemic infection of Bombyx mori. So, we should pay attention not only to N. bombycis, but also to other microsporidian (such as Nosema sp. HR) in sericulture in the future.

Baculovirus display systems have been utilized for cell-specific gene transfer, regenerative medicine, and as vaccine vectors. In particular, baculovirus particles displaying surface antigens have been used as vaccines against some parasites and viruses. In this study, Bombyx mori nucleopolyhedrovirus (BmNPV) particles displaying Neospora caninum antigens (NcSAG1, NcSRS2, and NcMIC3) purified from the hemolymph or fat body of silkworm larvae were prepared to vaccinate mice against N. caninum. Each antigen was expressed on the surface of BmNPV particles through glycoprotein 64 transmembrane and cytoplasmic domains. Antigen-specific antibody production was induced in mice by immunization with each recombinant BmNPV particle. NcMIC3-displaying BmNPV particles purified from the fat body induced a lower antibody titer than particles purified from the hemolymph. Antigen-specific IgG2a was predominantly produced in mice by immunization with NcSAG1-displaying BmNPV particles compared to IgG1, and induction of IFN-γ was dominant, indicating that antigen-displaying BmNPV particles can elicit a Th1 immune response in mice. Semi-quantitative PCR analysis revealed that immunization with each antigen-displaying BmNPV particle partially protected mice from cerebral N. caninum infection. These results suggest that antigen-displaying BmNPV particles can provide an alternative method of controlling neosporosis in cattle and represent a new generation of N. caninum vaccines.

Oviposition site-selection in insects is mediated through innate recognition templates (IRTs) tuned to specific chemical cues. These cues aid gravid insects in choosing suitable oviposition sites and may even enhance the fitness of their offspring by warding off predators and parasitoids. However, studies on the evolution of oviposition site-selection and cues instigating oviposition in domesticated insects remain elusive. Using the interaction between the silkmoth, Bombyx mori, and its host plant mulberry, Morus alba, as a model system, we demonstrate that centuries of domestication of silkmoth has not impaired its oviposition site-selection function. Silkmoths significantly preferred mulberry leaves to filter paper as oviposition sites. Oviposition assays with filter paper, filter paper treated with leaf volatiles and leaf alone proved that surface texture was not a significant criterion for oviposition site-selection, but volatile cues were. Oviposition assays with electrophysiologically active compounds from mulberry revealed that two of the volatiles, valencene and α-humulene, aided moths in choosing suitable oviposition sites and enhanced egg-laying significantly. Moreover, we show that generalist egg-parasitoids are strongly repelled by valencene and α-humulene. Our results demonstrate that IRTs tuned to cues that aid crucial functions like oviposition site-selection are less likely to be impaired even after centuries of domestication.

Infection of the commercially important silkworm, Bombyx mori by a tachnid parasitoid, Exorista bombycis induced activation of genes and cellular responses associated with apoptosis in integumental epithelial cells. Composite cellular profile showed initial autophagy, intermediate endoplasmic reticulum degranulation and deformed nucleus as well as later DNA fragmentation indicating apoptosis. Two cell death-associated proteins, autophagy 5-like (Atg5L) and apoptosis-inducing factor (AIF), in addition to caspase, are identified from the infected integumental epithelium through mass spectrometric analysis. Genes encoding these proteins showed age-dependent activation after the infection as revealed by quantitative expression analysis. Atg5 showed early upregulation in association with signs of autophagy whereas AIF showed late upregulation in association with DNA condensation and fragmentation. Expression of AIF showed negative correlation with that of Atg5 after the infection. On the other hand, in control, caspase expression showed positive correlation with AIF expression indicative of regulated expression in normal larval epithelium, which was absent after infection. Activation of Atg5, AIF and caspase genes in close association with different cell death events revealed the synchronized differential expression of apoptosis-associated genes in response to the macroparasitism. Enhanced expression of Atg5, AIF and caspase genes coupled with the appearance of cell death symptoms indicate parasitism-induced activation of genetic machinery to modulate cell death events in the epithelium, which was hither to unknown in invertebrate systems.

The activation of host response proteins against parasitic infection is dependent on the coregulation of immune gene expression. The infection of commercially important silkworm Bombyx mori through endoparasite Exorista bombycis enhanced host-response gene expression in integument early in the infection and was lowered asymptotically. Principal component analysis (PCA) showed heterogeneity while explaining ∼80 % variance among expression timings. PCA showed positive and negative correlation with gene expression and differentiated transcriptional timings, and revealed cross talk within the immune system. Pearson correlation analysis showed significant linear correlation (mean R (2) = >0.7; P < 0.004) between the expression of 16 pairs of genes in control, while the relation switched over to curvilinear due to parasitism. The genes showed pleiotropic interaction among them, with four genes each for prophenoloxidase activating enzyme (PPAE) and caspase. Besides, after parasitism, exclusive correlation of five gene pairs including PPAE-Spatzle pair (R (2) = 0.9; P < 0.011) was observed in the integument. In integument, the phenol oxidase (PO) activity showed a positive correlation with the tyrosine level (R (2) = 0.410; P < 0.002) and a curvilinear relation (R (2) = 0.745; P < 0.0002) with the expanding lysis area. The PO activity was positively correlated with BmToll expression and negatively correlated with paralytical peptide expression, revealing polygenic influence. Caspase expression was tightly regulated by signal genes in control integument, whereas they were deregulated after infection. Switchover from linear to curvilinear correlation and the appearance of new gene correlations in parasitized integument revealed deviation from gene coregulation, leading to impaired immune responses, characterized by lowered gene expression and varied phenotypic consequences.

Injection of culture supernatant of Serratia marcescens, a Gram-negative bacterium pathogenic to a wide range of host animals including insects and mammals, into the hemolymph of silkworm (Bombyx mori) larvae led to continuous flow of the hemolymph (blood of insects) from the injection site. The amount of hemolymph lost within 60 min reached 15-20% of the total larval weight. Using a bioassay with live silkworms, we purified Serralysin, a metalloprotease that requires divalent cations for its activity, as the factor responsible for the promotion of hemolymph bleeding from the culture supernatant of S. marcescens. Recombinant protein also induced hemolymph bleeding in silkworms. Moreover, the culture supernatant of an S. marcescens disruption mutant of the ser gene showed attenuated ability to promote hemolymph bleeding. In addition, this bleeding-promoting activity of the S. marcescens culture supernatant was attenuated by disruption of the wecA gene, which is involved in the biosynthesis of the lipopolysaccharide O-antigen. These findings suggest that Serralysin metalloprotease contributes to the pathogenesis of S. marcescens by inhibiting wound healing, which leads to a massive loss of hemolymph from silkworm larvae., (Copyright © 2014 Elsevier Inc. All rights reserved.)

In insects, the integument forms the primary barrier between the environment and internal milieu, but cellular and immune responses of the integumental epithelium to infection by micro- and macro-parasites are mostly unknown. We elucidated cellular and immune responses of the epithelium induced through infection by a dipteran endoparasitoid, Exorista bombycis in the economically important silkworm Bombyx mori. Degradative autophagic vacuoles, lamella-like bodies, a network of cytoplasmic channels with cellular cargo, and an RER network that opened to vacuoles were observed sequentially with increase in age after infection. This temporal sequence culminated in apoptosis, accompanied by the upregulation of the caspase gene and fragmentation of DNA. The infection significantly enhanced the tyrosine level and phenol oxidase activity in the integument. Proteomic analysis revealed enhanced expression of innate immunity components of toll and melanization pathways, cytokines, signaling molecules, chaperones, and proteolytic enzymes demonstrating diverse host responses. qPCR analysis revealed the upregulation of spatzle, BmToll, and NF kappa B transcription factors Dorsal and BmRel. NF kappa B inhibitor cactus showed diminished expression when Dorsal and BmRel were upregulated, revealing a negative correlation (R = (-)0.612). During melanization, prophenol oxidase 2 was expressed, a novel finding in integumental epithelium. The integument showed a low level of melanin metabolism and localized melanism in order to prevent the spreading of cytotoxic quinones. The gene-encoding proteolytic enzyme, beta-N-acetylglucosaminidase, was activated at 24 h post-infection, whereas chitinase, was activated at 96 h post-infection; however, most of the immune genes enhanced their expression in the early stages of infection. Thus the integument contributes to humoral immune responses that enhance resistance against macroparasite invasion.

Background: Microsporidian Nosema bombycis has received much attention because the pébrine disease of domesticated silkworms results in great economic losses in the silkworm industry. So far, no effective treatment could be found for pébrine. Compared to other known Nosema parasites, N. bombycis can unusually parasitize a broad range of hosts. To gain some insights into the underlying genetic mechanism of pathological ability and host range expansion in this parasite, a comparative genomic approach is conducted. The genome of two Nosema parasites, N. bombycis and N. antheraeae (an obligatory parasite to undomesticated silkworms Antheraea pernyi), were sequenced and compared with their distantly related species, N. ceranae (an obligatory parasite to honey bees)., Results: Our comparative genomics analysis show that the N. bombycis genome has greatly expanded due to the following three molecular mechanisms: 1) the proliferation of host-derived transposable elements, 2) the acquisition of many horizontally transferred genes from bacteria, and 3) the production of abundnant gene duplications. To our knowledge, duplicated genes derived not only from small-scale events (e.g., tandem duplications) but also from large-scale events (e.g., segmental duplications) have never been seen so abundant in any reported microsporidia genomes. Our relative dating analysis further indicated that these duplication events have arisen recently over very short evolutionary time. Furthermore, several duplicated genes involving in the cytotoxic metabolic pathway were found to undergo positive selection, suggestive of the role of duplicated genes on the adaptive evolution of pathogenic ability., Conclusions: Genome expansion is rarely considered as the evolutionary outcome acting on those highly reduced and compact parasitic microsporidian genomes. This study, for the first time, demonstrates that the parasitic genomes can expand, instead of shrink, through several common molecular mechanisms such as gene duplication, horizontal gene transfer, and transposable element expansion. We also showed that the duplicated genes can serve as raw materials for evolutionary innovations possibly contributing to the increase of pathologenic ability. Based on our research, we propose that duplicated genes of N. bombycis should be treated as primary targets for treatment designs against pébrine. The genome data and annotation information of N. bombycis and N.antheraeae were submitted to GenBank (Accession numbers ACJZ01000001 -ACJZ01003558).

Wolbachia are cytoplasmically inherited alpha-proteobacteria well known for inducing a variety of reproductive abnormalities in the diverse arthropod hosts they infect. Despite their obligate intracellular lifestyle which usually protects bacteria from phage infection, Wolbachia harbor a widespread temperate phage called WO. Evidences of horizontal phage transfers indicate that this phage could promote genetic exchanges between strains leading to evolutionary changes in the genomes of Wolbachia, and could be involved in the phenotypes these bacteria induced. In this study, we report the survey of Wolbachia and WO phage infections in 20 populations of the Uzifly Exorista sorbillans, a tachinid endoparasite of silkworm Bombyx mori, collected from different geographic regions of India. Previous studies demonstrated that Wolbachia is associated with positive reproductive fitness effects in this species. Polymerase chain reaction using the ftsZ gene encoding for a Wolbachia cell division protein and the orf7 capsid protein gene of the phage showed that all flies checked were infected by Wolbachia and its phage WO. Phylogenetic analyses based on the Wolbachia surface protein gene revealed 100% of double infections by the arthropod supergroups A and B. These results can serve as a valuable basis for understanding the evolution of Wolbachia bacteria and may provide information about the dynamics of Wolbachia-host associations. This knowledge could be exploited for the use of Wolbachia for effective control strategies of the Uzifly, a serious menace of the silkworm B. mori.

Life-cycle stages of the microsporidia Nosema bombycis, the pathogen causing silkworm pebrine, were separated and purified by an improved method of Percoll-gradient centrifugation. Soluble protein fractions of late sporoblasts (spore precursor cells) and mature spores were analysed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Protein spots were recovered from gels and analysed by mass spectrometry. The most abundant differential protein spot was identified by database search to be a hypothetical spore wall protein. Using immunoelectron microscopy, we demonstrated that HSWP5 is localized to the exospore of mature spores and renamed it as spore wall protein 5 (NbSWP5). Further spore phagocytosis assays indicated that NbSWP5 can protect spores from phagocytic uptake by cultured insect cells. This spore wall protein may function both for structural integrity and in modulating host cell invasion.

Several organophosphorus (OP) insecticides can selectively kill the silkworm maggot, Exorista sorbillans (Es) (Diptera: Tachinidae), while not obviously affecting the host (Bombyx mori) larvae, but the mechanism is not yet clear. In this study, the cDNA encoding an acetylcholinesterase (AChE) from the field Es was isolated. One point mutation (Gly353Ala) was identified. The Es-353G AChE and Es-353A AChE were expressed in baculovirus- insect cell system, respectively. The inhibition results showed that for eserine and Chlorpyrifos, Es-353A AChE was significantly less sensitive than Es-353G AChE. Meanwhile, comparison of the I(50) values of eserine, dichlorvos, Chlorpyrifos and omethoate of recombinant Es AChEs with its host (Bombyx mori) AChEs indicated that, both Es AChEs are more sensitive than B. mori AChEs. The results give an insight of the mechanism that some OP insecticides can selectively kills Es while without distinct effect on its host, B. mori.

Palmistichus elaeisis Delvare and LaSalle (Hymenoptera: Eulophidae) is a gregarious and polyphagous parasitoid mainly of Lepidoptera pupae. The objective of this paper as to study the developent of parasitoid on Bombyx mori L. (Lepidoptera: Bombycidae) pupae exposed to one, nine, 18, 27, 36, 45 or 54 female P. elaeisis, respectively. The females of the parasitoid remained in contact with pupae for 24 hours in glass tubes (14.0 x 2.2 cm), packed in a climatic chamber regulated at 25 +/- 2 degrees C, 70 +/- 10% relative humidity and photo phase of 12 hours. With the exception of density 1:1 (72.72%), in other densities parasitism was 100%. Adults of P. elaeisis did not emerge from pupae at densities of 1:1 and 9:1, but 100.0% of parasitoid emergence was observed at the density of 45:1 and 54.54% at 54:1. The duration of the life cycle of this parasitoid ranged from 20 to 28 days. P. elaeisis produced 49 to 589 descendants per pupa of B. mori. The sex ratio of P. elaeisis ranged from 0.93 +/- 0.01 to 0.97 +/- 0.01 without differences with 18, 27, 36, 45 and 54 females/host. This parasitoid should be reared with the density of 45 females per pupa of B. mori.

The mass rearing of parasitoids represents a fundamental stage for programmes of biological control. The progeny of the parasitoid Palmistichus elaeisis Delvare and LaSalle (Hymenoptera: Eulophidae) were evaluated on previously refrigerated pupae of Bombyx mori L. (Lepidoptera: Bombycidae). Forty-eight to 72 hours-old pupae of B. mori were stored at 10 degrees C for five, 10, 15 or 20 days and then exposed to parasitism by P. elaeisis females. This parasitoid showed shorter duration of the life cycle when reared on pupae of B. mori which were previously stored at 10 degrees C during 15 days. P. elaeisis parasitized 100% of the pupae of B. mori after storage at 10 degrees C during all periods with emergence of this parasitoid from 78 to 100% of these pupae. P. elaeisis had a higher number of progeny per pupa of B. mori stored for 15 days at 10 degrees C. Pupae of B. mori can be stored for 15 days at 10 degrees C before being used to rear P. elaeisis.

Random amplification of polymorphic DNA polymerase chain reaction (RAPD-PCR) and pathological, morphological and ultrastructural characterization were used to differentiate seven new microsporidian isolates infecting the mulberry silkworm, Bombyx mori. The pathogenicity observed was dose-dependent and differed from each of the microsporidian isolates; the NIK-4m was found to be more virulent than other isolates. However, all the isolates, except NIK-4m, showed heavy gonadal infection and vertical transmission in the infected silkworms. Differences in the spore shape ranging from oval to elongate were observed, and the polar filament has 8-16 coils arranged in one or two rows. Of the 80 decamer random primers tested, 50 generated reproducible RAPD profiles and yielded a total of 600 fragments, of which 594 were polymorphic (99%). Forty nine RAPD primers produced 179 unique genetic markers, whose presence or absence differed among the microsporidians, albeit with varied efficiency of polymorphism detection. The degree of band sharing was used to evaluate genetic distances between different microsporidian isolates and to construct a phylogenetic tree using Dice coefficients. Cluster analysis based on Dice coefficients resulted in the formation of one major cluster consisting of NIK-1s, NIAP-7g, NIK-2r and NIK-5d and NIK-4m in the other; while NIAP-6p was intermediate between these two. NIK-8b and NITN-9n were found to be entirely different from others. Reproducible RAPD patterns of all microsporidian isolates enabled us to differentiate the microsporidian isolates. The results demonstrate that besides ultrastructural studies, RAPD-PCR can be a useful and reliable tool to detect polymorphism, genetic relationships, and for the identification of the microsporidians. In addition, DNA fingerprints generated in this process have potential applications as diagnostic tools for identification of different microsporidia with considerable accuracy.

Nosema bombycis (Naegeli, 1857) is the causative agent of the "Pebrine disease" in silkworms. N. bombycis spores were found in silkworm samples originating from the Hatay, Bursa and Bilecik provinces. Clinical signs were observed in the infected silkworm larvae. Because of the transovarial transmission risk, all the infected silkworm colonies were destroyed.

Microsporidia are a group of intracellular parasites with an extremely compact genome and there is no confirmed evidence that retroelements are parasitised in these organisms. Using the dataset of 200,000 genomic shotgun reads of the silkworm pebrine Nosema bombycis, we have identified the eight complete N. bombycis long-terminal repeat retrotransposon (Nbr) elements. All of the Nbr elements are Ty3/gypsy members and have close relationships to Saccharomycetes long-terminal repeat retrotransposons identified previously, providing further evidence of their relationship to fungi. To explore the effect of retrotransposons in microsporidian genome evolution, their distribution was characterised by comparisons between two N. bombycis contigs containing the Nbr elements with the completed genome of the human parasite Encephalitozoon cuniculi, which is closely related to N. bombycis. The Nbr elements locate between or beside syntenic blocks, which are often clustered with other transposable-like sequences, indicating that they are associated with genome size variation and syntenic discontinuities. The ratios of the number of non-synonymous substitutions per non-synonymous site to the number of synonymous substitutions per synonymous site of the open reading frames among members of each of the eight Nbr families were estimated, which reveal the purifying selection acted on the N. bombycis long-terminal repeat retrotransposons. These results strongly suggest that retrotransposons play a major role in reorganization of the microsporidian genome and they might be active. The present study presents an initial characterization of some transposable elements in the N. bombycis genome and provides some insight into the evolutionary mechanism of microsporidian genomes.

Many arthropods harbour endosymbiotic bacteria of the genus Wolbachia. These endosymbionts are transmitted vertically from one generation to the next and are obligatory in several Dipterans that have been studied to date. These bacteria induce an array of reproductive isolation mechanisms that are implicated in pest management to evolutionary biology of respective hosts. The uzifly, Exorista sorbillans, a tachinid endoparasitoid of the silkworm, Bombyx mori L. (Lepidoptera: Bombycidae), causes enormous losses to the silk industry; now it is known that it harbours Wolbachia endobacteria. The elimination of Wolbachia by antibiotics interrupts embryogenesis and causes various reproductive conflicts such as (1) a reduction of fecundity of uninfected female, (2) cytoplasmic incompatibility in the uninfected females crossed with infected males, (3) genomic incompatibility in crosses between males and females from uninfected population, and (4) sex-ratio distortion in uninfected females irrespective of the presence of Wolbachia in males. These results suggest that the relationship of Wolbachia with its uzifly host is one of mutual symbiosis as it controls the reproductive physiology of its host., (Copyright 2005 Wiley-Liss, Inc.)

The uzifly, Exorista sorbillans (Diptera: Tachinidae), a parasitoid of the silkworm, Bombyx mori L. (Lepidoptera: Bombycidae), harbours Wolbachia (Rickettsia) endosymbionts. Administration of 0.05 mg/ml oxytetracycline to the adult uziflies removed Wolbachia endosymbionts and resulted in different reproductive disorders, such as i) reduction in fecundity of uninfected females, ii) cytoplasmic incompatibility in crosses between infected males and uninfected females, iii) sterility in the crosses between both males and females from uninfected populations, and iv) sex-ratio distortion in uninfected females irrespective of the presence of Wolbachia in males. However, tetracycline treatment did not have much effect on longevity of the uzifly. These results suggest that the interaction of Wolbachia with its uzifly host is one of mutual symbiosis as it controls the reproductive physiology of its hosts.

Exorista sorbillans is a tachinid endoparasitoid of silkworm, Bombyx mori, and is globally known as uzi. It causes economic injury to the cocoon crop in silkworm cultivating areas of India, except those above 400 m above mean sea level (AMSL) in the foothills of the Himalayas (Darjeeling). It is reported that the sericulture tract of south India became infected with this pest only since 1980 through an accidental transportation of cocoons from West Bengal. To ascertain whether the genome of this parasitoid is differentiating into discrete gene pools in contrasting geo-climatic conditions, molecular profiling of four populations (Es (Annatapur), Es(Ramanagaram), Es (Channapatna) and Es(Kodathi) from south India and Es(Murshidabad) from Murshidabad, West Bengal was undertaken with 13 ISSR, 3 RAPD and six non-random primers designed from various repeat sequences of B. mori . MANOVA indicated significance for the Roy's largest root estimate (55.4; F =18.47; p = 0.002) for the variability contributed by the replication. Further, hierarchical clustering done on the basis of Euclidean distance matrix and Nei's unbiased Phylip clustering put Es(Murshidabad) at the maximum distance from those of south India and 29 markers could also be identified which significantly differentiateEs(Murshidabad) from others. However, Nei's statistics for gene diversity in sub-populations reveal considerably high gene-flow (3.44 and 2.51) among the populations around Bangalore. The gene-flow between Es(Murshidabad) and other population is lowest but cannot be ignored. The comparison of endosymbiont specific 16SrRNA and fts Z gene (partial) sequences through clustalW (gcgMSF) revealed a closer relationship of Es(Murshidabad) with Es(Annatapur) and Es (Ramanagaram) and is not congruent with the relationships discussed above. The significance of this maiden study with a tachinid fly-pest is discussed in the context of understanding the diversification of Uzi fly-pest and also establishing this pest as a relevant biological material for studying microevolution in future.

This study is the first report on the genetic characterization and relationships among different microsporidia infecting the silkworm, Bombyx mori, using inter simple sequence repeat PCR (ISSR-PCR) analysis. Six different microsporidians were distinguished through molecular DNA typing using ISSR-PCR. Thus, ISSR-PCR analysis can be a powerful tool to detect polymorphisms and identify microsporidians, which are difficult to study with microscopy because of their extremely small size. Of the 100 ISSR primers tested, only 28 primers had reproducibility and high polymorphism (93%). A total of 24 ISSR primers produced 55 unique genetic markers, which could be used to differentiate the microsporidians from each other. Among the 28 SSRs tested, the most abundant were (CA)n, (GA)n, and (GT)n repeats. The degree of band sharing was used to evaluate genetic similarity between different microsporidian isolates and to construct a phylogenetic tree using Jaccard's similarity coefficient. The results indicate that the DNA profiles based on ISSR markers can be used as diagnostic tools to identify different microsporidia with considerable accuracy. In addition, the small subunit ribosomal RNA (SSU-rRNA) sequence gene was amplified, cloned, and sequenced from each of the 6 microsporidian isolates. These sequences were compared with 20 other microsporidian SSU-rRNA sequences to develop a phylogenetic tree for the microsporidia isolated from the silkworms. This method was found to be useful in establishing the phylogenetic relationships among the different microsporidians isolated from silkworms. Of the 6 microsporidian isolates, NIK-1s revealed an SSU-rRNA gene sequence similar to Nosema bombycis, indicating that NIK-1s is similar to N. bombycis; the remaining 5 isolates, which differed from each other and from N. bombycis, were considered to be different variants belonging to the species N. bombycis.

The pathogenicity, mode of transmission, tissue specificity of infection and the small subunit rRNA (SSU-rRNA) gene sequences of the three new microsporidian isolates from the silkworm Bombyx mori were studied. Out of the three, NIK-2r revealed life cycle features and SSU-rRNA gene sequence similar to Nosema bombycis, suggesting that it is N. bombycis. The other two, NIK-4m and NIK-3h, differed from each other as well as from N. bombycis. NIK-4m was highly pathogenic and did not show any vertical transmission, in accordance with the apparent lack of gonadal infection, whereas NIK-3h was less pathogenic and vertical transmission was not detected but could not be excluded. Phylogenetic analysis based on SSU-rRNA gene sequence placed NIK-3h and NIK-4m in a distinct clade that included almost all the Vairimorpha species and Nosema species that infect lepidopteran and non-lepidopteran hosts, while NIK-2r was included in a clade containing almost all the Nosema isolates that infect only lepidopteran hosts. Thus, we have presented molecular evidence that one of the three isolates is in fact the type species N. bombycis, while the other two isolates are Vairimorpha spp. There was distinct separation of microsporidian isolates infecting only lepidopteran hosts and those infecting lepidopteran and non-lepidopteran hosts, reflecting possible co-evolution of hosts and microsporidian isolates.

Using a mixture of several PCR primers, we evaluated whether multiprimer PCR is practically useful for the early and simultaneous detection of several kinds of microsporidia that cause silkworm pebrine. When genomic DNA extracted from silkworm eggs infected with Nosema bombycis was used as the DNA template, the specific DNA sequences were amplified by multiprimer PCR. In addition, similar results were obtained even when genomic DNA extracted from silkworms infected with N. bombycis was used as the DNA template. These findings suggest that multiprimer PCR using several primers designed for this study is practically useful for pebrine inspection of silkworm eggs.

The entomogenous fungus Nomuraea rileyi reportedly secretes a proteinaceous substance inhibiting larval molt and metamorphosis in the silkworm Bombyx mori. We studied the possibility that N. rileyi controls B. mori development by inactivating hemolymph molting hormone, ecdysteroids. Incubation of ecdysone (E) and 20-hydroxyecdysone (20E) in fungal-conditioned medium resulted in their rapid modification into products with longer retention times in reverse-phase HPLC. Each modified product from E and 20E was purified by HPLC, and identified by NMR as 22-dehydroecdysone and 22-dehydro-20-hydroxyecdysone. Some other ecdysteroids with a hydroxyl group at position C22 were also modified. Injection of the fungal-conditioned medium into Bombyx mori larvae in the mid-4th instar inhibited larval molt but induced precocious pupal metamorphosis, and its injection into 5th instar larvae just after gut purge blocked pupal metamorphosis. In hemolymph of injected larvae, E and 20E disappeared and, in turn, 22-dehydroecdysone and 22-dehydro-20-hydroxyecdysone accumulated. These results indicate that N. rileyi secretes a specific enzyme that oxidizes the hydroxyl group at position C22 of hemolymph ecdysteroids and prevents molting in B. mori larvae., (Copyright 2003 Wiley-Liss, Inc.)

Nuclotide sequence (1205 bp) of small subunit ribosomal RNA (SSUrDNA) of a microsporidium isolated from Pieris rapae L. (abbr:MPr) was specifically amplified by polymerase chain reaction (PCR). Another fragment of 657 bp downstream of MPr SSUrDNA 3'end was amplified with two other primers. Within this 657 bp fragment, the putative 3'terminus of MPr SSUrDNA and the extreme 5' of large subunit ribosomal RNA gene. (LSUrDNA) were identified, which situated at base 145, 146-186 and 187, respectively. Then the full sequence of MPr ssurDNA is 1245 bp. Its GC content was also nearly 34%. The ITS region (internal transcribed spacer), positioned between the ssu and LSUrRNA genes, was found to be 41 bp in length. The LSUrDNA 580r region of MPr is 470 bp, longer than 437 bp of Nosema apis, 447 bp of Nosema algerae. The secondary structure of MPr SSUrRNA was constructed. These analyses of MPr rRNA gene contributed to the somewhat limited microsporidian taxonomic classification based on morphology.

A microsporidium (called as Bab-M) was isolated from Barathra brassicae L. captured from suburban vegetable plot of Guang Zhou. The spores were long-ovoid in shape and 4.02 +/- 0.36 microns x 1.99 +/- 0.36 microns in size. Immunologically the microsporidium shared spore surface specific antigen(s) with N. bombycis. The ultrastructure and life cycle of Bab-M were similar to that of N. b.. The rate of transovarian transmission was high. The initial conclusion was that Bab-M should be referred to as Nosema bombycis, but there was variation between them.

Latex agglutination assay based on monoclonal antibodies (MCAs) described in this communication may be useful for detection of Pebrine infection in silkworm. Four murine MCAs were produced against Nosema bombycis spore. In ELISA all 4 MCAs (IgM isotype) reacted with alkali treated Nosema spores and to variable extent with acetone precipitated surface protein. However, MA-310 and MA-542 showed a low degree of cross reactivity with BmNPV. In contrast, MA-503 and MA-515 were devoid of reactivity with BmNPV, B. thuringiensis, S. marcescens, Azotobactor, Rhizobium and normal hemolymph protein in ELISA. Latex beads sensitized with a combination of MA-503 and MA-515 (50 micrograms each per ml of 0.4% latex beads) could detect 1 x 10(5) Nosema spores per test. Sensitization of the latex beads with the cocktail of these two MCAs through protein-A bridge further led to a 10-fold increase in the sensitivity (1 x 10(4) spores/test) of the assay. No agglutination was observed in presence of BmNPV, Rhizobium, Azotobactor, E. coli, B. thuringiensis, S. marcescens and normal hemolymph protein indicating the specificity of the test. The results obtained by latex agglutination assay on hemolymph samples of infected as well as normal larvae collected from field, II instar larvae infected in the laboratory and from infected mother moth revealed 100% correlation with results by microscopic examination.

Biological interrelations between the agent of microsporidiosis of the silkworm and local species of insects were studied. 7 species of Lepidoptera (Agrotis segetum, Chloridea obsoleta, Laphygma exigua, Plusia gamma, Pieris brassicae, P. rapae, Lymantria dispar) were infected with the suspension of Nosema bombycis spores. Results have shown that 6 species of insects are susceptible to Nosema bombycis excluding only the gipsy moth. So, N. bombycis can be considered to be a parasite with a wide range of hosts.